Antisense antiviral compound and method for treating arenavirus infection

ABSTRACT

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Arenaviridae family and in the treatment of a viral infection. The compounds are particularly useful in the treatment of Arenavirus infection in a mammal. The antisense antiviral compounds are substantially uncharged morpholino oligonucleotides have a sequence of 12-40 subunits, including at least 12 subunits having a targeting sequence that is complementary to a region associated with viral RNA sequences within a 19 nucleotide region of the 5′-terminal regions of the viral RNA, viral complementary RNA and/or mRNA identified by SEQ ID NO:1.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/715,572 filed Mar. 7, 2007, now pending, which application claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/780,228 filed Mar. 7, 2006; which applications are incorporatedherein by reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made, in part, with U.S. Government support underGrant Nos. T32 NS-041219, AI-050840, and AI-065359, awarded by theNational Institutes of Health to co-inventors M. Buchmeier and B.Neuman. As to the rights of M. Buchmeier and B. Neuman, or any assigneethereof, in this invention, the U.S. Government has certain rights inthe invention.

FIELD OF THE INVENTION

This invention relates to antisense oligonucleotide compounds for use intreating an arenavirus infection and antiviral treatment methodsemploying the compounds.

REFERENCES

-   Agrawal, S., S. H. Mayrand, et al. (1990). “Site-specific excision    from RNA by RNase H and mixed-phosphate-backbone    oligodeoxynucleotides.” Proc Natl Acad Sci USA 87(4):1401-5.-   Barton, L. L., M. B. Mets, et al. (2002). “Lymphocytic    choriomeningitis virus: emerging fetal teratogen.” Am J Obstet    Gynecol 187(6):1715-6.-   Blommers, M. J., U. Pieles, et al. (1994). “An approach to the    structure determination of nucleic acid analogues hybridized to RNA.    NMR studies of a duplex between 2′-OMe RNA and an oligonucleotide    containing a single amide backbone modification.” Nucleic Acids Res    22(20):4187-94.-   Bonham, M. A., S. Brown, et al. (1995). “An assessment of the    antisense properties of RNase H-competent and steric-blocking    oligomers.” Nucleic Acids Res 23(7):1197-203.-   Boudvillain, M., M. Guerin, et al. (1997). “Transplatin-modified    oligo(2′-O-methyl ribonucleotide)s: a new tool for selective    modulation of gene expression.” Biochemistry 36(10):2925-31.-   Cross, C. W., J. S. Rice, et al. (1997). “Solution structure of an    RNA×DNA hybrid duplex containing a 3′-thioformacetal linker and an    RNA A-tract.” Biochemistry 36(14):4096-107.-   Ding, D., S. M. Grayaznov, et al. (1996). “An    oligodeoxyribonucleotide N3′-->P5′ phosphoramidate duplex forms an    A-type helix in solution.” Nucleic Acids Res 24(2):354-60.-   Egholm, M., O. Buchardt, et al. (1993). “PNA hybridizes to    complementary oligonucleotides obeying the Watson-Crick    hydrogen-bonding rules.” Nature 365(6446):566-8.-   Felgner, P. L., T. R. Gadek, et al. (1987). “Lipofection: a highly    efficient, lipid-mediated DNA-transfection procedure.” Proc Natl    Acad Sci USA 84(21):7413-7.-   Gait, M. J., A. S. Jones, et al. (1974). “Synthetic-analogues of    polynucleotides XII. Synthesis of thymidine derivatives containing    an oxyacetamido- or an oxyformamido-linkage instead of a    phosphodiester group.” J Chem Soc [Perkin 1] 0(14)1684-6.-   Gee, J. E., I. Robbins, et al. (1998). “Assessment of high-affinity    hybridization, RNase H cleavage, and covalent linkage in translation    arrest by antisense oligonucleotides.” Antisense Nucleic Acid Drug    Dev 8(2):103-11.-   Knipe, D. M., P. M. Howley, et al. (2001). Fields Virology,    Lippincott.-   Lesnikowski, Z. J., M. Jaworska, et al. (1990). “Octa(thymidine    methanephosphonates) of partially defined stereochemistry: synthesis    and effect of chirality at phosphorus on binding to    pentadecadeoxyriboadenylic acid.” Nucleic Acids Res 18(8):2109-15.-   Mertes, M. P. and E. A. Coats (1969). “Synthesis of carbonate    analogs of dinucleosides. 3′-Thymidinyl 5′-thymidinyl carbonate,    3′-thymidinyl 5′-(5-fluoro-2′-deoxyuridinyl) carbonate, and    3′-(5-fluoro-2′-deoxyuridinyl) 5′-thymidinyl carbonate.” J Med Chem    12(1):154-7.-   Meyer, B. J., J. C. de la Torre, et al. (2002). “Arenaviruses:    genomic RNAs, transcription, and replication.” Curr Top Microbiol    Immunol 262:139-57.-   Moulton, H. M., M. H. Nelson, et al. (2004). “Cellular uptake of    antisense morpholino oligomers conjugated to arginine-rich    peptides.” Bioconjug Chem 15(2):290-9.-   Nelson, M. H., D. A. Stein, et al. (2005). “Arginine-rich peptide    conjugation to morpholino oligomers: effects on antisense activity    and specificity.” Bioconjug Chem 16(4):959-66.-   Polyak, S. J., S. Zheng, et al. (1995). “5′ termini of Pichinde    arenavirus S RNAs and mRNAs contain nontemplated nucleotides.” J    Virol 69(5):3211-5.-   Strauss, J. H. and E. G. Strauss (2002). Viruses and Human Disease.    San Diego, Academic Press.-   Summerton, J. and D. Weller (1997). “Morpholino antisense oligomers:    design, preparation, and properties.” Antisense Nucleic Acid Drug    Dev 7(3):187-95.

BACKGROUND OF THE INVENTION

The family Arenaviridae contains a single genus, Arenavirus, consistingof at least 16 currently recognized species that can be divided into OldWorld and New World viruses as shown in Table 1. Because of theirassociation with individual rodent species, arenavirus species arerestricted to that of their host. Rodents that have been distributedwidely by humans also spread their associated virus as exemplified bythe prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV).LCMV is an Old World virus that is associated with the house mouse Musdomesticus and Mus musculus and is found throughout Europe and theAmericas.

The most significant Arenavirus species with regards to pathogenic humaninfectious disease are the Old World viruses LCMV and Lassa virus (LASV)and the New World viruses Junin virus (JUNV) also known as ArgentineHemorrhagic Fever virus (AHF), Machupo virus (MACV) also known asBolivian Hemorrhagic Fever virus (BHF), Guanarito virus (GTOV) alsoknown as Venezuelan Hemorrhagic Fever virus (VHF), Sabia virus (SABV)and Whitewater Arroyo virus (WWAV).

LCMV is less virulent for man than the other Arenaviruses and casesusually present as a viral meningitis although deeper neurologicinvolvement is evident in a minority of cases, perhaps 10% or fewer innaturally observed outbreaks. Encephalitis has been diagnosed in 5% to34% of hospitalized patients with documented LCMV. Full recovery isusual, although occasional deaths do occur (Knipe, Howley et al. 2001).A more significant risk to humans is the threat of fetal LCMVinfections. It is becoming increasingly apparent that LCMV is animportant cause of fetal abnormalities in the United States (Barton,Mets et al. 2002).

Lassa virus is endemic to West Africa and causes between 100,000 to300,000 cases a year (Strauss and Strauss 2002) and the mortality ofhospitalized cases is 15-20% (Knipe, Howley et al. 2001). Fatal Lassavirus infection is a relentless disease with the progression of symptomsculminating in the onset of shock and death. Clinical manifestationsinclude aseptic meningitis, encephalitis, global encephalopathy withseizures, and more subtle neurologic problems. Lassa virus is also knownto cause unusually high fetal mortality.

The New World Arenaviruses are very important disease agents that causelarge outbreaks of hemorrhagic fever with high mortality rates. Thenumber of cases is increasing with development and expanding populationsthat bring humans in closer association with the rodent reservoirs.

An effective attenuated virus vaccine against Junin virus (AHF) has beendeveloped and is used widely in populations at risk of infection.However, no vaccines are in use for the other Arenaviruses. Passiveimmunotherapy against some Arenaviruses has shown promise but thisapproach is complicated due to limited availability and the need totreat with large volumes of plasma, typically two to three units.

The only existing antiviral drug used to treat infections by the virusesdescribed above is the guanosine analog ribavarin which has shown to bemoderately effective against a limited subset of the arenavirus species.Ribavarin penetrates poorly into the cerebral spinal fluid which limitsits potential as an LCMV antiviral drug.

All Arenaviruses form stable, infectious aerosols and have beenimportant causes of laboratory infections and deaths and consequentlyare manipulated under BSL-4 containment. The potential for many of theseviruses to be used as agents of bioterrorism or biowarfare is widelyaccepted and as a result LCMV, JUNV, MACV, GTOV and LASV are listed asCategory A Pathogens by the National Institute of Allergy and InfectiousDisease (NIAID).

Thus, there remains a need for a more effective antiviral therapyagainst several members of the Arenaviridae family.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a method of inhibiting viralinfection in mammalian cells by a species in the Arenaviridae family.The method includes the steps of exposing the cells to an antisenseoligonucleotide compound, thereby to form a heteroduplex structure (i)composed of the virus' vRNA, vcRNA and/or mRNA strands and theoligonucleotide compound, and (ii) characterized by a Tm of dissociationof at least 45° C. The oligonucleotide compound is characterized by:

(i) a substantially uncharged, nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases, and

(iv) having a targeting sequence of at least 12 subunits complementaryto SEQ ID NO:1 in either the vRNA, vcRNA and/or mRNA strands of thevirus

The compound to which the host cells are exposed may be composed ofmorpholino subunits and phosphorus-containing intersubunit linkagesjoining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon ofan adjacent subunit. The morpholino subunits may be joined byphosphorodiamidate linkages having the structure:

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino.

The oligonucleotide compound to which the cells are exposed may have asequence complementary to SEQ ID NO:1, such as one of the sequencesidentified by SEQ ID NOS:2-5. The compound may be conjugated to anarginine-rich polypeptide effective to promote uptake of the compoundinto infected host cells. Exemplary arginine-rich polypeptides have oneof the sequences identified as SEQ ID NOS:6-12.

For use in treating a mammalian subject infected by a virus of theArenaviridae family, the compound is administered to the subject in apharmaceutically effective amount. Compound administration may becontinued until a significant reduction in viral infection or thesymptoms thereof is observed. The subject may be treated with a secondanti-viral compound before, after, or during treatment with theoligonucleotide compound.

For use in treating a mammalian subject at risk of infection by a virusof the Arenaviridae family, the compound is administered to the subjectin an amount effective to inhibit infection of subject host cells by thevirus.

In another aspect, the invention includes an oligonucleotide compoundfor use in inhibiting viral infection in mammalian cells by a virus ofthe Arenaviridae family. The compound is characterized by:

(i) a substantially uncharged, nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases,

(iv) having a targeting sequence of at least 12 subunits complementaryto SEQ ID NO:1; and

(v) capable of binding to the virus' vRNA, vcRNA and/or mRNA strands toform a heteroduplex structure having by a Tm of dissociation of at least45° C.

The compound may be composed of morpholino subunits andphosphorus-containing intersubunit linkages joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.The morpholino subunits may be joined by phosphorodiamidate linkageshaving the structure:

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino.

The oligonucleotide compound may have one of the sequences identified bySEQ ID NOS:2-5. The compound may be conjugated to an arginine-richpolypeptide effective to promote uptake of the compound into infectedhost cells. Exemplary arginine-rich polypeptides have one of thesequences identified as SEQ ID NOS:6-12.

The compound may be formulated in combination with another anti-viralcompound.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the repeating subunit segment of exemplary morpholinooligonucleotides, designated A through D.

FIGS. 2A-2G show examples of uncharged linkage types in oligonucleotideanalogs. FIG. 2H is an example of a charged, cationic linkage.

FIG. 3 shows the sequence conservation across a broad spectrum ofArenaviruses for the 19 nucleotide 5′ terminal region of both the L- andS-segment RNA strands represented by the combined sequence identified bySEQ ID NO:1.

FIG. 4 is a schematic diagram showing the ambisense coding strategy forthe S- and L-segments of a typical Arenavirus, the genome organizationof the S- and L-segments and the replicative and transcriptional eventsin the viral life-cycle.

FIG. 5 shows the viral titer reduction when JUNV-infected Vero E6 cellsare treated with antisense P-PMO targeting the 5′-termini of vRNA/vcRNA.

FIG. 6 is a series of photomicrographs showing the reduction of viralcytopathic effects when JUNV-infected Vero E6 cells are treated withantisense P-PMO targeting the 5′-termini of vRNA/vcRNA.

FIG. 7 is a series of photomicrographs showing the reduction ofTCRV-induced cytophathic effects when infected Vero E6 cells are treatedwith antisense P-PMO targeting the 5-termini of vRNA/vcRNA.

FIG. 8 shows a graph depicting the viral titer reduction (VTR) whenLCMV-infected Vero E6 cells are treated with P-PMO targeting the5′-termini of vRNA/vcRNA.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

The terms “oligonucleotide analog” refers to an oligonucleotide having(i) a modified backbone structure, e.g., a backbone other than thestandard phosphodiester linkage found in natural oligo- andpolynucleotides, and (ii) optionally, modified sugar moieties, e.g.,morpholino moieties rather than ribose or deoxyribose moieties. Theanalog supports bases capable of hydrogen bonding by Watson-Crick basepairing to standard polynucleotide bases, where the analog backbonepresents the bases in a manner to permit such hydrogen bonding in asequence-specific fashion between the oligonucleotide analog moleculeand bases in a standard polynucleotide (e.g., single-stranded RNA orsingle-stranded DNA). Preferred analogs are those having a substantiallyuncharged, phosphorus containing backbone. An oligonucleotide analog isalso referred to herein as an oligonucleotide or oligonucleotidecompound or oligonucleotide analog compound.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 60-100%, typically at least 80% of its linkages,are uncharged at physiological pH, and contain a single phosphorousatom. The analog contains between 8 and 40 subunits, typically about8-25 subunits, and preferably about 12 to 25 subunits. The analog mayhave exact sequence complementarity to the target sequence or nearcomplementarity, as defined below.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

A “morpholino oligonucleotide analog” or “morpholino oligonucleotidecompound” is an oligonucleotide analog composed of morpholino subunitstructures of the form shown in FIGS. 1A-1D where (i) the structures arelinked together by phosphorus-containing linkages, one to three atomslong, joining the morpholino nitrogen of one subunit to the 5′ exocycliccarbon of an adjacent subunit, and (ii) P_(i) and P_(j) are purine orpyrimidine base-pairing moieties effective to bind, by base-specifichydrogen bonding, to a base in a polynucleotide. The purine orpyrimidine base-pairing moiety is typically adenine, cytosine, guanine,uracil or thymine. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in U.S. Pat. Nos.5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and5,506,337, all of which are incorporated herein by reference.

The subunit and linkage shown in FIG. 1B are used for six-atomrepeating-unit backbones, as shown in FIG. 1B (where the six atomsinclude: a morpholino nitrogen, the connected phosphorus atom, the atom(usually oxygen) linking the phosphorus atom to the 5′ exocyclic carbon,the 5′ exocyclic carbon, and two carbon atoms of the next morpholinoring). In these structures, the atom Y₁ linking the 5′ exocyclicmorpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusis any stable group which does not interfere with base-specific hydrogenbonding. Preferred X groups include fluoro, alkyl, alkoxy, thioalkoxy,and alkyl amino, including cyclic amines, all of which can be variouslysubstituted, as long as base-specific bonding is not disrupted. Alkyl,alkoxy and thioalkoxy preferably include 1-6 carbon atoms. Alkyl aminopreferably refers to lower alkyl (C₁ to C₆) substitution, and cyclicamines are preferably 5- to 7-membered nitrogen heterocycles optionallycontaining 1-2 additional heteroatoms selected from oxygen, nitrogen,and sulfur. Z is sulfur or oxygen, and is preferably oxygen.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 1B,where X=NH₂, NHR, or NR₂ (where R is lower alkyl, preferably methyl),Y=O, and Z=O, and P_(i) and P_(j) are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide. Also preferred are structures having an alternatephosphorodiamidate linkage, where, in FIG. 1B, X=lower alkoxy, such asmethoxy or ethoxy, Y=NH or NR, where R is lower alkyl, and Z=O.

The term “substituted”, particularly with respect to an alkyl, alkoxy,thioalkoxy, or alkylamino group, refers to replacement of a hydrogenatom on carbon with a heteroatom-containing substituent, such as, forexample, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino,imino, oxo (keto), nitro, cyano, or various acids or esters such ascarboxylic, sulfonic, or phosphonic. It may also refer to replacement ofa hydrogen atom on a heteroatom (such as an amine hydrogen) with analkyl, carbonyl or other carbon containing group.

As used herein, the term “Arenavirus” refers to one or more viralspecies belonging to the Arenaviridae family and specifically virusescategorized as either Old World Arenaviruses or New World Arenaviruseswithin the Arenavirus genus.

As used herein, the term “target” refers to a viral RNA region, andspecifically, to a region identified by SEQ ID NO:1 at the 5′-termini ofeither the viral RNA (vRNA), viral complementary RNA (vcRNA) or viralmRNA of a member of the Arenaviridae described herein.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide analog is directed, that is, the sequence towhich the oligonucleotide analog will hybridize by Watson-Crick basepairing of a complementary sequence.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the analog compound may be complementaryto the target sequence. For example, in an analog having 20 bases, only12-14 may be targeting sequences. Typically, the targeting sequence isformed of contiguous bases in the analog, but may alternatively beformed of non-contiguous sequences that when placed together, e.g., fromopposite ends of the analog, constitute sequence that spans the targetsequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the present invention haveat most one mismatch with the target sequence out of 10 nucleotides, andpreferably at most one mismatch out of 20. Alternatively, the antisenseoligomers employed have at least 90% sequence homology, and preferablyat least 95% sequence homology, with the exemplary targeting sequencesas designated herein. For purposes of complementary binding to an RNAtarget, and as discussed below, a guanine base may be complementary toeither an adenine or uracil RNA base.

An oligonucleotide analog “specifically hybridizes” to a targetpolynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a T_(m) substantially greater than 45°C., preferably at least 50° C., and typically 60° C.-80° C. or higher.Such hybridization preferably corresponds to stringent hybridizationconditions. At a given ionic strength and pH, the T_(m) is thetemperature at which 50% of a target sequence hybridizes to acomplementary polynucleotide. Again, such hybridization may occur with“near” or “substantial” complementary of the antisense oligomer to thetarget sequence, as well as with exact complementarity.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

A “heteroduplex” refers to a duplex between an oligonculeotide analogand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAse H, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

A “base-specific intracellular binding event involving a target RNA”refers to the specific binding of an oligonucleotide analog to a targetRNA sequence inside a cell. The base specificity of such binding issequence specific. For example, a single-stranded polynucleotide canspecifically bind to a single-stranded polynucleotide that iscomplementary in sequence.

An “effective amount” of an antisense oligomer, targeted against aninfecting Arenavirus, is an amount effective to reduce the rate ofreplication of the infecting virus, and/or viral load, and/or symptomsassociated with the viral infection.

As used herein, the term “body fluid” encompasses a variety of sampletypes obtained from a subject including, urine, saliva, plasma, blood,spinal fluid, or other sample of biological origin, such as skin cellsor dermal debris, and may refer to cells or cell fragments suspendedtherein, or the liquid medium and its solutes.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Treatment” of an individual or a cell is any type of interventionprovided as a means to alter the natural course of the individual orcell. Treatment includes, but is not limited to, administration of e.g.,a pharmaceutical composition, and may be performed eitherprophylactically, or subsequent to the initiation of a pathologic eventor contact with an etiologic agent. The related term “improvedtherapeutic outcome” relative to a patient diagnosed as infected with aparticular virus, refers to a slowing or diminution in the growth ofvirus, or viral load, or detectable symptoms associated with infectionby that particular virus.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, the oligonucleotide analog preferably has a substantiallyuncharged backbone, as defined below. Alternatively, the antisensecompound may be formulated in a complexed form, such as an agent havingan anionic backbone complexed with cationic lipids or liposomes, whichcan be taken into cells by an endocytotic mechanism. The analog also maybe conjugated, e.g., at its 5′ or 3′ end, to an arginine-rich peptide,e.g., a portion of the HIV TAT protein, polyarginine, or combinations ofarginine and other amino acids including the non-natural amino acids6-aminohexanoic acid and beta-alanine. Exemplary arginine-rich deliverypeptides are listed as SEQ ID NOS:6-12. These exemplary arginine-richdelivery peptides facilitate transport into the target host cell asdescribed (Moulton, Nelson et al. 2004; Nelson, Stein et al. 2005).

Rules for the selection of targeting sequences capable of inhibitingreplication of Arenaviruses are discussed below.

II. Targeted Viruses

The present invention is based on the discovery that effectiveinhibition of members of the Arenaviridae family can be achieved withantisense oligonucleotide analog compounds that (i) target the regionidentified by SEQ ID NO:1 at the 5′ terminus of both the S-L-segment RNAstrands, and (ii) have physical and pharmacokinetic features which alloweffective interaction between the antisense compound and the viruswithin host cells. In one aspect, the oligomers can be used in treatinga mammalian subject infected with the virus.

The invention targets RNA viruses that are members of the Arenaviridaefamily including members of the Arenavirus genera, the sole genus of theArenaviridae family. Table 1 is an exemplary list of viruses targeted bythe invention as organized by their Old World or New World Arenavirusclassification. Various physical, morphological, and biologicalcharacteristics of members of the Arenaviridae family can be found, forexample, in Textbook of Human Virology, R. Belshe, ed., 2^(nd) Edition,Mosby, 1991, at the Universal Virus Database of the InternationalCommittee on Taxonomy of Viruses(http://www.ncbi.nlm.nih.gov/ICTVdb/index.htm) and in human virologytextbooks (e.g., see (Knipe, Howley et al. 2001) and (Strauss andStrauss 2002)). Some of the key biological characteristics of theArenaviridae family of viruses are described below.

TABLE 1 Targeted Viruses of the Invention Family Genus VirusArenaviridae Arenavirus Old World Arenaviruses Lassa virus (LASV)Lymphocytic choriomeningitis virus (LCMV) Mopeia virus (MOPV) New WorldArenaviruses Guanarito virus (GTOV) Junin virus (JUNV) Machupo virus(MACV) Pichinide virus (PICV) Pirital virus (PIRV) Sabia virus (SABV)Tacaribe virus (TCRV) Whitewater Arroyo virus (WWAV)

Genomic Organization of Arenaviruses

All Arenaviruses are enveloped and have a bi-segmented RNA with a uniqueambisense genomic organization (Knipe, Howley et al. 2001; Meyer, de laTorre et al. 2002). The genome of Arenaviruses consists of twosingle-stranded RNA segments designated S (small) and L (large). Invirions, the molar ratio of S- to L-segment RNAs is roughly 2:1. Thecomplete S-segment RNA sequence has been determined for severalarenaviruses and ranges from 3,366 to 3,535 nucleotides. The completeL-segment RNA sequence has also been determined for several arenavirusesand ranges from 7,102 to 7,279 nucleotides. The 3′ terminal sequences ofthe S and L RNA segments are identical at 17 of the last 19 nucleotides(for the S-segment it is 5″-GCCUAGGAUCCACUGUGC-3′, and for L-segment itis 5″-GCCUAGGAUCCUCGGUGCG-3′). These terminal sequences are conservedamong all known arenaviruses as shown in FIG. 3. The 5′-terminal 19 or20 nucleotides at the beginning of each genomic RNA are imperfectlycomplementary with each corresponding 3′ end. Because of thiscomplementarity, the 3′ and 5′ termini are thought to base-pair and formpanhandle structures. An additional G residue, which would notparticipate in base-pairing with a corresponding 3′-terminal base, hasbeen detected on the 5′ end of some genomic RNAs (Polyak, Zheng et al.1995). In these cases, the panhandle structures would not contain flushends, for there would be a single G overhang on the 5′ side.

Replication of the infecting virion or viral RNA (vRNA) to form anantigenomic, viral-complementary RNA (vcRNA) strand occurs in theinfected cell (FIG. 4). Both the vRNA and vcRNA encode complementarymRNAs and because of that Arenaviruses are classified as ambisense RNAviruses as opposed to either negative- or positive-sense RNA viruses.The ambisense orientation of viral genes are on both the L- andS-segments (FIG. 4). The NP and polymerase genes reside at the 3′ end ofthe S and L vRNA segments, respectively, and are encoded in theconventional negative sense (i.e., they are expressed throughtranscription of vRNA or genome-complementary mRNAs). The genes locatedat the 5′ end of the S and L vRNA segments, GPC and Z, respectively, areencoded in mRNA sense but there is no evidence that they are translateddirectly from genomic vRNA. These genes are expressed instead throughtranscription of genomic-sense mRNAs from antigenomes (i.e., the vcRNA),full-length complementary copies of genomic vRNAs that function asreplicative intermediates (FIG. 4).

GenBank reference entries for exemplary viral nucleic acid sequencesrepresenting Arenavirus vRNA are listed in Table 2 below. The nucleotidesequence numbers in Table 2 are derived from the Genbank reference forthe vcRNA. It will be appreciated that these sequence references areonly illustrative of other sequences in the Arenaviridae family, as maybe available from available gene-sequence databases or literature orpatent resources.

Antisense Oligomer Targets in the Arenavirus Genome

Table 2 lists the antisense targets for a 19-base sequence correspondingto nucleotides 1-19 or 2-20 and contained in the 5′-terminal region ofboth the S- and L-segments of the listed Arenaviruses. All the viruseslisted in Table 2 are human isolates The target sequence (SEQ ID NO:1)is 5′-CGCACMGDGGATCCTAGGC-3′ where the International Union of Pure andApplied Chemistry (IUPAC) nomenclature for incompletely specified basesare used in the description of the sequence (i.e., “M” for either C or Aand “D” for either A, G or T).

An important feature of the present invention is the high degree ofsequence conservation between Arenaviruses at the 5′ terminus of thevRNA and vcRNA, as shown in FIG. 3. The points where the antisenseoligomers of the present invention can exert their antiviral effects areshown schematically in FIG. 4 as indicated by an “X”. The targetsinclude the 5′ termini of either the S- or L-segment vRNA or vcRNAstrands or the 5′ termini of any of the four viral mRNAs. As such, theoligomers potentially disrupt viral replication, transcription ortranslation of viral RNA species.

The prototypic member of the Arenaviridae family is lymphocyticchoriomeningitis virus (LCMV). Table 2 lists the corresponding targetregions in a number of clinically relevant Arenaviruses and thosepresent in the NCBI Reference Sequence database. Both the ReferenceSequence Number (Ref. No.) and the GenBank Accession number (GB No.) areprovided. The target homologies for the target region across severalArenaviruses is shown in FIG. 3. The target sequence identified as SEQID NO:1 represents a combined target sequence, where the positionsindicated by the letter “M” may be either C or A and “D” is either A, Gor T.

TABLE 2 Exemplary Human Arenavirus Nucleic Acid Target Sequences SEQVirus Ref. No. GB No. Segment Region ID NO LASV NC_004296 J04324 S 1-191 LASV NC_004297 U73034 L 1-19 1 LCMV NC_004294 M20869 S 1-19 1 LCMVNC_004291 J04331 L 1-19 1 MOPV NC_006575 AY772170 S 1-19 1 MOPVNC_006574 AY772169 L 1-19 1 GTOV NC_005077 AY129247 S 1-19 1 GTOVNC_005082 AY358024 L 1-19 1 JUNV NC_005081 AY358023 S 1-19 1 JUNVNC_005080 AY358022 L 1-19 1 MACV NC_005078 AY129248 S 1-19 1 MACVNC_005079 AY358021 L 1-19 1 PICV NC_006447 K02734 S 1-19 1 PICVNC_006439 AF427517 L 1-19 1 PIRV NC_005894 AF485262 S 1-19 1 PIRVNC_005897 AY494081 L 1-19 1 SABV NC_006317 U41071 S 1-19 1 SABVNC_006313 AY358026 L 1-19 1 TCRV NC_004293 M20304 S 1-19 1 TCRVNC_004292 J04340 L 1-19 1

Targeting sequences are designed to hybridize to a region of the targetsequence as listed in Table 3. Selected targeting sequences can be madeshorter, e.g., 12 bases, or longer, e.g., 40 bases, and include a smallnumber of mismatches, as long as the sequence is sufficientlycomplementary to hybridize with the target, and forms with theArenavirus vRNA/vcRNA or mRNA strands, a heteroduplex having a Tm of 45°C. or greater.

More generally, the degree of complementarity between the target andtargeting sequence is sufficient to form a stable duplex. The region ofcomplementarity of the antisense oligomers with the target RNA sequencemay be as short as 8-11 bases, but is preferably 12-15 bases or more,e.g. 12-20 bases, or 12-25 bases. An antisense oligomer of about 14-15bases is generally long enough to have a unique complementary sequencein the viral genome. In addition, a minimum length of complementarybases may be required to achieve the requisite binding T_(m), asdiscussed below.

Oligomers as long as 40 bases may be suitable, where at least theminimum number of bases, e.g., 8-11, preferably 12-15 bases, arecomplementary to the target sequence. In general, however, facilitatedor active uptake in cells is optimized at oligomer lengths less thanabout 30, preferably less than 25, and more preferably 20 or fewerbases. For PMO oligomers, described further below, an optimum balance ofbinding stability and uptake generally occurs at lengths of 14-24 bases.

The oligomer may be 100% complementary to the viral nucleic acid targetsequence, or it may include mismatches, e.g., to accommodate variants orincrease broad reactivity to different viral species, as long as aheteroduplex formed between the oligomer and viral nucleic acid targetsequence is sufficiently stable to withstand the action of cellularnucleases and other modes of degradation which may occur in vivo.Oligomer backbones which are less susceptible to cleavage by nucleasesare discussed below. Mismatches, if present, are less destabilizingtoward the end regions of the hybrid duplex than in the middle. Thenumber of mismatches allowed will depend on the length of the oligomer,the percentage of G:C base pairs in the duplex, and the position of themismatch(es) in the duplex, according to well understood principles ofduplex stability. Although such an antisense oligomer is not necessarily100% complementary to the viral nucleic acid target sequence, it iseffective to stably and specifically bind to the target sequence, suchthat a biological activity of the nucleic acid target, e.g., expressionof viral protein(s) and/or replication of viral RNA, is modulated.

The oligomer may also incorporate guanine bases in place of adenine whenthe target nucleotide is a uracil residue. This is useful when thetarget sequence varies across different viral species and the variationat any given nucleotide residue is either cytosine or uracil. Byutilizing guanine in the targeting oligomer at the position ofvariability, the well-known ability of guanine to base pair with uracil(termed C/U:G base pairing) can be exploited. By incorporating guanineat these locations, a single oligomer can effectively target a widerrange of RNA target variability. Similarly, variation at a givennucleotide can be accommodated by inclusion of inosine in the targetingoligomer. Inosine is capable of forming base pairs with any nucleotidein the complementary strand. An example of this is shown in Table 3 asSEQ ID NOS:5 and 6. Although the target sequence shown in FIG. 3 andlisted in Table 2 contains T for thymidine, which is the convention forsequence listings, it will be appreciated that because Arenaviruses areRNA viruses, the T residues refer to uracil.

The stability of the duplex formed between the oligomer and the targetsequence is a function of the binding T_(m) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(m) of an antisensecompound with respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques. Methods Enzymol. Vol. 154 pp. 94-107. Each antisenseoligomer should have a binding T_(m), with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 50° C. T_(m)'s in the range 60-80° C. or greaterare preferred. According to well known principles, the T_(m) of anoligomer compound, with respect to a complementary-based RNA hybrid, canbe increased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high T_(m) (50° C. or greater) at a length of 20bases or less are generally preferred over those requiring greater than20 bases for high T_(m) values.

Table 3 below shows exemplary targeting sequences, in a 5′-to-3′orientation, that are complementary to a broad spectrum of Arenaviruses.The targeting sequences listed below in Table 3 provide a collection oftargeting sequences from which targeting sequences may be selected,according to the general class rules discussed above. The CL-trm, LS-trmand SS-trm targeting oligomers (SEQ ID NOS:2-4, respectively) were usedin experiments conducted in support of the invention as described in theExamples and were designed to target specifically Junin-Candid-1. Asshown below, the targeting sequences represented by SEQ ID NOS:5 and 6incorporate inosine (“I”) at two positions of sequence variabilityacross a broad range of Arenavirus species.

TABLE 3 Exemplary Antisense Oligomer Targeting Sequences Target GenBankTargeting SEQ. PMO Nucleotides Acc. No. Antisense Oligomer (5′ to 3′) IDNO. CL-trm 1-20 NC_005080 CGC CTA GGA TCC CCG GTG CG 2 LS-trm 1-21NC_005080 CGC CTA GGA TCC CCG GTG CGC 3 SS-trm 1-20 NC_005081 GCC TAGGAT CCA CTG TGC GC 4 PanCL 1-19 N/A GCC TAG GAT CCI CIG TGC G 5 PanLS1-20 N/A CGC CTA GGA TCC ICI GTG CG 6

III. Antisense Oligonucleotide Analog Compounds

A. Properties

As detailed above, the antisense oligonucleotide analog compound has abase sequence targeting a region that includes one or more of thefollowing; 1) the 5′ untranslated region of either the vRNA, vcRNA ormRNA and; 2) the 5′-terminal 19 bases of the vRNA, vcRNA or mRNA. Inaddition, the oligomer is able to effectively target infecting viruses,when administered to a host cell, e.g. in an infected mammalian subject.This requirement is met when the oligomer compound (a) has the abilityto be actively taken up by mammalian cells, and (b) once taken up, forma duplex with the target RNA with a T_(m) greater than about 45° C.

As will be described below, the ability to be taken up by cells requiresthat the oligomer backbone be substantially uncharged, and, preferably,that the oligomer structure is recognized as a substrate for active orfacilitated transport across the cell membrane. The ability of theoligomer to form a stable duplex with the target RNA will also depend onthe oligomer backbone, as well as factors noted above, the length anddegree of complementarity of the antisense oligomer with respect to thetarget, the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Below are disclosed methods for testing any given, substantiallyuncharged backbone for its ability to meet these requirements.

B. Active or Facilitated Uptake by Cells

The antisense compound may be taken up by host cells by facilitated oractive transport across the host cell membrane if administered in free(non-complexed) form, or by an endocytotic mechanism if administered incomplexed form.

In the case where the agent is administered in free form, the antisensecompound should be substantially uncharged, meaning that a majority ofits intersubunit linkages are uncharged at physiological pH. Experimentscarried out in support of the invention indicate that a small number ofnet charges, e.g., 1-2 for a 15- to 20-mer oligomer, can in fact enhancecellular uptake of certain oligomers with substantially unchargedbackbones. The charges may be carried on the oligomer itself, e.g., inthe backbone linkages, or may be terminal charged-group appendages.Preferably, the number of charged linkages is no more than one chargedlinkage per four uncharged linkages. More preferably, the number is nomore than one charged linkage per ten, or no more than one per twenty,uncharged linkages. In one embodiment, the oligomer is fully uncharged.

An oligomer may also contain both negatively and positively chargedbackbone linkages, as long as opposing charges are present inapproximately equal number. Preferably, the oligomer does not includeruns of more than 3-5 consecutive subunits of either charge. Forexample, the oligomer may have a given number of anionic linkages, e.g.phosphorothioate or N3′P5′ phosphoramidate linkages, and a comparablenumber of cationic linkages, such as N,N-diethylenediaminephosphoramidates (Dagle, Littig et al. 2000). The net charge ispreferably neutral or at most 1-2 net charges per oligomer.

The antisense compound may also be administered in complexed form, wherethe complexing agent is typically a polymer, e.g., a cationic lipid,polypeptide, or non-biological cationic polymer, having an oppositecharge to any net charge on the antisense compound. Methods of formingcomplexes, including bilayer complexes, between anionic oligonucleotidesand cationic lipid or other polymer components, are well known. Forexample, the liposomal composition Lipofectin (Feigner, Gadek et al.1987), containing the cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. After administration, the complex is taken up by cells through anendocytotic mechanism, typically involving particle encapsulation inendosomal bodies.

The antisense compound may also be administered in conjugated form withan arginine-rich peptide linked covalently to the 5′ or 3′ end of theantisense oligomer. The peptide is typically 8-16 amino acids andconsists of a mixture of arginine, and other amino acids includingphenyalanine, cysteine, beta-alanine and 6-aminohexanoic acid. Exemplaryarginine-rich delivery peptides are described in the Sequence Listingtable as SEQ ID NOS:7-11. The use of arginine-rich peptide-PMOconjugates can be used to enhance cellular uptake of the antisenseoligomer (See, e.g. (Moulton, Nelson et al. 2004; Nelson, Stein et al.2005).

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., antisense oligonucleotides: a newtherapeutic principle, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747.

Alternatively, and according to another aspect of the invention, therequisite properties of oligomers with any given backbone can beconfirmed by a simple in vivo test, in which a labeled compound isadministered to an animal, and a body fluid sample, taken from theanimal several hours after the oligomer is administered, assayed for thepresence of heteroduplex with target RNA. This method is detailed insubsection D below.

C. Substantial Resistance to RNaseH

Two general mechanisms have been proposed to account for inhibition ofexpression by antisense oligonucleotides. (See e.g., (Agrawal, Mayrandet al. 1990; Bonham, Brown et al. 1995; Boudvillain, Guerin et al.1997). In the first, a heteroduplex formed between the oligonucleotideand the viral RNA acts as a substrate for RNaseH, leading to cleavage ofthe viral RNA. Oligonucleotides belonging, or proposed to belong, tothis class include phosphorothioates, phosphotriesters, andphosphodiesters (unmodified “natural” oligonucleotides). Such compoundsexpose the viral RNA in an oligomer:RNA duplex structure to hydrolysisby RNaseH, and therefore loss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and are believed to act bysterically blocking target RNA nucleocytoplasmic transport, splicing ortranslation. This class includes methylphosphonates (Toulme et al.,1996), morpholino oligonucleotides, peptide nucleic acids (PNA's),certain 2′-O-allyl or 2′-O-alkyl modified oligonucleotides (Bonham,Brown et al. 1995), and N3→P5′ phosphoramidates (Ding, Grayaznov et al.1996; Gee, Robbins et al. 1998).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under a standard assay conditions, as described in Stein etal. After exposure to RNaseH, the presence or absence of intact duplexcan be monitored by gel electrophoresis or mass spectrometry.

D. In Vivo Uptake

In accordance with another aspect of the invention, there is provided asimple, rapid test for confirming that a given antisense oligomer typeprovides the required characteristics noted above, namely, high T_(m),ability to be actively taken up by the host cells, and substantialresistance to RNaseH. This method is based on the discovery that aproperly designed antisense compound will form a stable heteroduplexwith the complementary portion of the viral RNA target when administeredto a mammalian subject, and the heteroduplex subsequently appears in theurine (or other body fluid). Details of this method are also given inco-owned U.S. patent application Ser. No. 09/736,920, entitled“Non-Invasive Method for Detecting Target RNA” (Non-Invasive Method),the disclosure of which is incorporated herein by reference.

Briefly, a test oligomer containing a backbone to be evaluated, having abase sequence targeted against a known RNA, is injected into a mammaliansubject. The antisense oligomer may be directed against anyintracellular RNA, including a host RNA or the RNA of an infectingvirus. Several hours (typically 8-72) after administration, the urine isassayed for the presence of the antisense-RNA heteroduplex. Ifheteroduplex is detected, the backbone is suitable for use in theantisense oligomers of the present invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

When the antisense oligomer is complementary to a virus-specific regionof the viral genome (such as those regions of Arenavirus RNA, asdescribed above) the method can be used to detect the presence of agiven Arenavirus virus, or reduction in the amount of virus during atreatment method.

E. Exemplary Oligomer Backbones

Examples of nonionic linkages that may be used in oligonucleotideanalogs are shown in FIGS. 2A-2G. In these figures, B represents apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide,preferably selected from adenine, cytosine, guanine, thymidine anduracil. Suitable backbone structures include carbonate (2A, R=O) andcarbamate (2A, R=NH₂) linkages (Mertes and Coats 1969; Gait, Jones etal. 1974); alkyl phosphonate and phosphotriester linkages (2B, R=alkylor —O-alkyl) (Lesnikowski, Jaworska et al. 1990); amide linkages (2C)(Blommers, Pieles et al. 1994); sulfone and sulfonamide linkages (2D,R₁, R₂=CH₂); and a thioformacetyl linkage (2E) (Cross, Rice et al.1997). The latter is reported to have enhanced duplex and triplexstability with respect to phosphorothioate antisense compounds (Cross,Rice et al. 1997). Also reported are the3′-methylene-N-methylhydroxyamino compounds of structure 2F.

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligonucleotides obeying Watson-Crick base-pairingrules, and mimic DNA in terms of base pair recognition (Egholm, Buchardtet al. 1993). The backbone of PNAs are formed by peptide bonds ratherthan phosphodiester bonds, making them well-suited for antisenseapplications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNAduplexes which exhibit greater than normal thermal stability. PNAs arenot recognized by nucleases or proteases.

A preferred oligomer structure employs morpholino-based subunits bearingbase-pairing moieties, joined by uncharged linkages, as described above.Especially preferred is a substantially unchargedphosphorodiamidate-linked morpholino oligomer, such as illustrated inFIGS. 1A-1D. Morpholino oligonucleotides, including antisense oligomers,are detailed, for example, in co-owned U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and5,506,337, all of which are expressly incorporated by reference herein.

Important properties of the morpholino-based subunits include: theability to be linked in a oligomeric form by stable, uncharged backbonelinkages; the ability to support a nucleotide base (e.g. adenine,cytosine, guanine, thymidine, uracil or inosine) such that the polymerformed can hybridize with a complementary-base target nucleic acid,including target RNA, with high T_(m), even with oligomers as short as10-14 bases; the ability of the oligomer to be actively transported intomammalian cells; and the ability of the oligomer:RNA heteroduplex toresist RNAse degradation.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the α-morpholino subunit types are also shown in FIGS.1A-1D, each linked by an uncharged, phosphorus-containing subunitlinkage. FIG. 1A shows a phosphorus-containing linkage which forms thefive atom repeating-unit backbone, where the morpholino rings are linkedby a 1-atom phosphoamide linkage. FIG. 1B shows a linkage which producesa 6-atom repeating-unit backbone. In this structure, the atom Y linkingthe 5′ morpholino carbon to the phosphorus group may be sulfur,nitrogen, carbon or, preferably, oxygen. The X moiety pendant from thephosphorus may be fluorine, an alkyl or substituted alkyl, an alkoxy orsubstituted alkoxy, a thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures, such as morpholines or piperidines. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms. The Z moieties aresulfur or oxygen, and are preferably oxygen.

The linkages shown in FIGS. 1C and 1D are designed for 7-atomunit-length backbones. In Structure 1C, the X moiety is as in Structure1B, and the moiety Y may be methylene, sulfur, or, preferably, oxygen.In Structure 1D, the X and Y moieties are as in Structure 1B.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 1B,where X=NH₂ or N(CH₃)₂, Y=O, and Z=O and in FIG. 2G.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages, more preferably up to about 1 per every 10uncharged linkages. Therefore a small number of charged linkages, e.g.charged phosphoramidate or phosphorothioate, may also be incorporatedinto the oligomers. An exemplary cationic linkage structure is shown inFIG. 2H.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above. Insome cases, it may be desirable to add additional chemical moieties tothe antisense compound, e.g. to enhance pharmacokinetics or tofacilitate capture or detection of the compound. Such a moiety may becovalently attached, typically to a terminus of the oligomer, accordingto standard synthetic methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 monomeric subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an antisense oligomer, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by a subject without undesirable sideeffects.

IV. Inhibition of Arenavirus Viral Replication

The antisense compounds detailed above are useful in inhibitingreplication of single-stranded, ambi-sense RNA viruses of theArenaviridae family. In one embodiment, such inhibition is effective intreating infection of a host animal by these viruses. Accordingly, themethod comprises, in one embodiment, exposing a mammalian cell infectedwith the virus with an oligonucleotide antisense compound effective toinhibit the replication of the specific virus. In this embodiment, thecells are exposed to the compound either in vitro or in vivo, where themethod is used in the latter case to treat a mammalian subject, e.g.,human or domestic animal, infected with a given virus. It iscontemplated that the antisense oligonucleotide arrests the growth ofthe RNA virus in the host. The RNA virus may be decreased in number oreliminated with little or no detrimental effect on the normal growth ordevelopment of the host.

In the present invention as described in the Examples,phosphorodiamidate morpholino oligomers (PMOs), designed to hybridize tospecific regions of the Junin-Candid Arenavirus 5′-terminal regions, areevaluated for their ability to inhibit viral replication and/orinduction of cytopathic effects when used against three differentArenaviruses, LCMV, JUNV and TCRV. The target region is highly conservedwithin the Arenaviridae family. The PMOs described herein will targetmost, if not all, Arenavirus species because of the high degree ofhomology between viral species at the target (SEQ ID NO:1) as shown inFIG. 3.

A. Identification of the Infective Agent

The specific virus causing the infection can be determined by methodsknown in the art, e.g. serological, genotyping, or cultural methods, orby methods employing the antisense oligomers of the present invention.

Serological identification employs a viral sample or culture isolatedfrom a biological specimen, e.g., stool, urine, cerebrospinal fluid,blood, nasopharyngeal secretions, etc., of the subject. Immunoassay forthe detection of virus is generally carried out by methods routinelyemployed by those of skill in the art, e.g., ELISA or Western blot. Inaddition, monoclonal antibodies specific to particular viral strains orspecies are often commercially available.

Culture methods may be used to isolate and identify particular types ofvirus, by employing techniques including, but not limited to, comparingcharacteristics such as rates of growth and morphology under variousculture conditions.

Genotyping methods include polymerase chain reaction (PCR) methods usinggenotype-specific primers or genomic sequencing of viral nucleic acidobtained from the infected individual.

Another method for identifying the viral infective agent in an infectedsubject employs one or more antisense oligomers targeting broad familiesand/or genera of viruses. Sequences targeting any characteristic viralRNA can be used. The desired target sequences are preferably (i) commonto broad virus families/genera, and (ii) not found in humans.Characteristic nucleic acid sequences for a large number of infectiousviruses are available in public databases, and may serve as the basisfor the design of specific oligomers.

For each plurality of oligomers, the following steps are carried out:(a) the oligomer(s) are administered to the subject; (b) at a selectedtime after said administering, a body fluid sample is obtained from thesubject; and (c) the sample is assayed for the presence of anuclease-resistant heteroduplex comprising the antisense oligomer and acomplementary portion of the viral genome. Steps (a)-(c) are carried forat least one such oligomer, or as many as is necessary to identify thevirus or family of viruses. Oligomers can be administered and assayedsequentially or, more conveniently, concurrently. The virus isidentified based on the presence (or absence) of a heteroduplexcomprising the antisense oligomer and a complementary portion of theviral genome of the given known virus or family of viruses.

Preferably, a first group of oligomers, targeting broad families, isutilized first, followed by selected oligomers complementary to specificgenera and/or species and/or strains within the broad family/genusthereby identified. This second group of oligomers includes targetingsequences directed to specific genera and/or species and/or strainswithin a broad family/genus. Several different second oligomercollections, i.e. one for each broad virus family/genus tested in thefirst stage, are generally provided. Sequences are selected which are(i) specific for the individual genus/species/strains being tested and(ii) not found in humans.

B. Administration of the Antisense Oligomer

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. In accordance with the invention,routes of antisense oligomer delivery include, but are not limited to,various systemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery. The appropriate routemay be determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of a antisense oligomer in the treatment of a viralinfection of the skin is topical delivery, while delivery of a antisenseoligomer for the treatment of a viral respiratory infection is byinhalation. The oligomer may also be delivered directly to the site ofviral infection, or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically acceptable. Such a composition may include anyof a variety of standard pharmaceutically accepted carriers employed bythose of ordinary skill in the art. Examples include, but are notlimited to, saline, phosphate buffered saline (PBS), water, aqueousethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. The choice of suitable physiologicallyacceptable carrier will vary dependent upon the chosen mode ofadministration.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligomer into cells. (See, e.g., Williams, S. A., Leukemia10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res. 23:119, 1994;Uhlmann et al., antisense oligonucleotides: a new therapeutic principle,Chemical Reviews, Volume 90, No. 4, pages 544-584, 1990; Gregoriadis,G., Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp.287-341, Academic Press, 1979). As described above, the use ofarginine-rich cellular delivery peptides conjugated to the antisenseoligomer may also be used. Hydrogels may also be used as vehicles forantisense oligomer administration, for example, as described in WO93/01286. Alternatively, the oligomers may be administered inmicrospheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C. H., J.Biol. Chem. 262:4429-4432, 1987). Alternatively, the use of gas-filledmicrobubbles complexed with the antisense oligomers can enhance deliveryto target tissues, as described in U.S. Pat. No. 6,245,747.

Sustained release compositions may also be used. These may includesemipermeable polymeric matrices in the form of shaped articles such asfilms or microcapsules.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a localized or systemic viral infection. Thecondition of a patient may also dictate prophylactic administration ofan antisense oligomer of the invention, e.g. in the case of a patientwho (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; or (4) is about to undergo or has recentlyundergone surgery. In one preferred embodiment, the oligomer is aphosphorodiamidate morpholino oligomer, contained in a pharmaceuticallyacceptable carrier, and is delivered orally. In another preferredembodiment, the oligomer is a phosphorodiamidate morpholino oligomer,contained in a pharmaceutically acceptable carrier, and is deliveredintravenously (i.v.).

The antisense compound is generally administered in an amount and mannereffective to result in a peak blood concentration of at least 200-400 nMantisense oligomer. Typically, one or more doses of antisense oligomerare administered, generally at regular intervals, for a period of aboutone to two weeks. Preferred doses for oral administration are from about1-100 mg oligomer per 70 kg. In some cases, doses of greater than 100 mgoligomer/patient may be necessary. For i.v. administration, preferreddoses are from about 0.5 mg to 100 mg oligomer per 70 kg. The antisenseoligomer may be administered at regular intervals for a short timeperiod, e.g., daily for two weeks or less. However, in some cases theoligomer is administered intermittently over a longer period of time.Administration may be followed by, or concurrent with, administration ofan antibiotic or other therapeutic treatment. The treatment regimen maybe adjusted (dose, frequency, route, etc.) as indicated, based on theresults of immunoassays, other biochemical tests and physiologicalexamination of the subject under treatment.

C. Monitoring of Treatment

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will often require monitoring by testsappropriate to the particular type of viral infection under treatment,and corresponding adjustments in the dose or treatment regimen, in orderto achieve an optimal therapeutic outcome. Treatment may be monitored,e.g., by general indicators of infection, such as complete blood count(CBC), nucleic acid detection methods, immunodiagnostic tests, viralculture, or detection of heteroduplex.

The efficacy of an in vivo administered antisense oligomer of theinvention in inhibiting or eliminating the growth of one or more typesof RNA virus may be determined from biological samples (tissue, blood,urine etc.) taken from a subject prior to, during and subsequent toadministration of the antisense oligomer. Assays of such samples include(1) monitoring the presence or absence of heteroduplex formation withtarget and non-target sequences, using procedures known to those skilledin the art, e.g., an electrophoretic gel mobility assay; (2) monitoringthe amount of viral protein production, as determined by standardtechniques such as ELISA or Western blotting, or (3) measuring theeffect on viral titer, e.g. by the method of Spearman-Karber. (See, forexample, Pari, G. S. et al., Antimicrob. Agents and Chemotherapy39(5):1157-1161, 1995; Anderson, K. P. et al., Antimicrob. Agents andChemotherapy 40(9):2004-2011, 1996, Cottral, G. E. (ed) in: Manual ofStandard Methods for Veterinary Microbiology, pp. 60-93, 1978).

A preferred method of monitoring the efficacy of the antisense oligomertreatment is by detection of the antisense-RNA heteroduplex. At selectedtime(s) after antisense oligomer administration, a body fluid iscollected for detecting the presence and/or measuring the level ofheteroduplex species in the sample. Typically, the body fluid sample iscollected 3-24 hours after administration, preferably about 6-24 hoursafter administering. As indicated above, the body fluid sample may beurine, saliva, plasma, blood, spinal fluid, or other liquid sample ofbiological origin, and may include cells or cell fragments suspendedtherein, or the liquid medium and its solutes. The amount of samplecollected is typically in the 0.1 to 10 ml range, preferably about 1 mlof less.

The sample may be treated to remove unwanted components and/or to treatthe heteroduplex species in the sample to remove unwanted ssRNA overhangregions, e.g. by treatment with RNase. It is, of course, particularlyimportant to remove overhang where heteroduplex detection relies on sizeseparation, e.g., electrophoresis of mass spectroscopy.

A variety of methods are available for removing unwanted components fromthe sample. For example, since the heteroduplex has a net negativecharge, electrophoretic or ion exchange techniques can be used toseparate the heteroduplex from neutral or positively charged material.The sample may also be contacted with a solid support having asurface-bound antibody or other agent specifically able to bind theheteroduplex. After washing the support to remove unbound material, theheteroduplex can be released in substantially purified form for furtheranalysis, e.g., by electrophoresis, mass spectroscopy or immunoassay.

V. Examples

The following examples illustrate but are not intended in any way tolimit the invention.

A. Materials and Methods

All peptides are custom synthesized by Global Peptide Services (Ft.Collins, Colo.) or at AVI BioPharma (Corvallis, Oreg.) and purifiedto >90% purity. Phosphorodiamidate morpholino oligomers (PMOs) aresynthesized at AVI BioPharma in accordance with known methods, asdescribed, for example, in (Summerton and Weller 1997) and U.S. Pat. No.5,185,444.

For the examples described below, PMO oligomers are conjugated at the 5′end with an arginine-rich peptide, P007 or (RXR)₄XB-PMO (where R isarginine, X is 6-aminohexanoic acid and B is beta-alanine), to enhancecellular uptake as described (U.S. Patent Application 60/466,703 and(Moulton, Nelson et al. 2004; Nelson, Stein et al. 2005). This peptideis also called P007 and listed as SEQ ID NO:8 in the Sequence Listingtable. PMOs conjugated to a delivery peptide are called P-PMOs.

Cells and Viruses

Vero-E6 cells were cultured in DMEM containing 10% fetal bovine serum,0.01 M HEPES, penicillin and streptomycin for general growth andmaintenance, or in serum-free medium (VP-SFM; Invitrogen) supplementedwith L-glutamine, penicillin and streptomycin during P-PMO studies.Infectious stocks of Junin-Candid#1 (JUNV), Tacaribe virus (TCRV) andlymphocytic choriomeningitis virus-Armstrong (LCMV) were prepared onVero-E6 cells.

Virus Growth and Titer Reduction Assays

Vero-E6 cells were seeded at a density of 5×10⁵ cells per 25 cm² tissueculture flask and allowed to adhere overnight at 37° C., 5% CO₂. Cellswere pre-treated with 1 ml VP-SFM containing treatment for 6 h, exceptwhere stated, as in time-of-addition and time-of-removal experiments.Cells were inoculated with JUNV or LCMV at a multiplicity of 0.1PFU/cell and placed at 37° C. for 1 h. Inoculum was removed and replacedwith fresh VP-SFM with or without P-PMO treatment. Cells in the JUNV andLCMV experiments included with this submission were treated with 15 μMP-PMO in VP-SFM. Cell culture medium was collected, stored and replacedwith fresh medium at designated timepoints. Virus in cell culturesupernatants was titrated by plaque assay. Cells were fixed with 10%formaldehyde in phosphate buffered saline and stained with 0.1% crystalviolet to assess cytopathic effects (CPE).

Plaque Assay

For arenavirus plaque assays, Vero-E6 cells were seeded in 12-welltissue culture plates at 2×10⁵ cells per well and allowed to adhereovernight at 37° C., 5% CO₂. Culture medium was removed and replacedwith 0.5 ml of inoculum. Cells were treated as specified, and a 2% fetalbovine serum, 0.7% agarose overlay was applied 1 h after inoculation.After 5 d, cells were fixed with 10% formaldehyde in phosphate bufferedsaline, agarose plugs were removed and cells were stained with 0.1%crystal violet.

Example 1 Inhibition of Junin-Candid-1 Virus (JUNV) in Tissue Culturewith PMOs that Target the 5′ Termini of the vRNA/vcRNA Strands

The antiviral activity of Junin-Candid-1-specific PMOs were determinedby measuring viral replication and cytopathic effects in JUNV-infectedcells. The tests are performed on Vero-E6 cells as described above. Cellmonolayers (12-well plates) are seeded 16 to 20 hours prior to treatmentwith PMO or infection with virus and pretreated pretreated with 1 ml of15 micromolar P-PMO in serum-free culture medium for 6 h beforeinoculation. Cells were infected at a multiplicity of infection (MOI) of0.01 plaque forming units per cell (PFU/cell). Culture medium wascollected 96 hours after inoculation and infectious virus was titratedby plaque assay. After collection of the culture medium, cells werefixed with 25% neutral buffered formalin and stained with crystal violetto visualize multinucleate syncytia, a measure of viral-inducedcytopathic effects (CPE).

P-PMOs directed at the precise 5′-termini of the vRNA and vcRNA strands,which share a common 19 nt terminal repeat sequence, demonstratedsignificant inhibitory effects on JUNV replication. As shown in FIG. 5,the terminus-binding P-PMOs (CL-trm, LS-trm and SS-trm, SEQ ID NOS:2-4,respectively) reduced viral titer by 100 to 10000-fold 4 d afterinoculation compared to viral titers from untreated and randomizedP-PMO-treated cells (DSCR) (FIG. 5). The most effective P-PMOs (CL-trm,LS-trm and SS-trm, SEQ ID NOS:2-4, respectively) also reduced CPE, asmeasured by syncytium formation which results from expression of theviral-encoded GP-C protein on the cell surface, to undetectable levelsas shown in FIG. 6.

Example 2 Inhibition of Tacaribe Virus (TCRV) Cytopathic Effects inTissue Culture with PMOs that Target the 5′ Termini of the vRNA/vcRNAStrands

Antiviral activity against Tacaribe virus (TCRV, another New Worldarenavirus) was measured by observation of CPE in tissue cultureexperiments similar to those described for JUNV in Example 1 above. Thesame series of P007-conjugated P-PMO were selected for these analysesand used to treat Vero-E6 cells under the same conditions described inExample 1 above. Six hours post-treatment with PMO, cells were infectedwith Tacaribe virus at an MOI of 0.01 pfu/cell. Photomicrographs weretaken 96 hours post-infection as shown in FIG. 7. A significantreduction in CPE was observed with the same three P-PMOs (CL-trm, LS-trmand SS-trm, SEQ ID NOS:2-4, respectively) compared to the scramblecontrol P-PMO (DSCR) treated and untreated TCRV-infected cultures asshown in FIG. 7.

Example 3 Inhibition of Lymphocytic Choriomeningitis Virus (LCMV) inTissue Culture with PMOs that Target the 5′ Termini of the vRNA/vcRNAStrands

The most effective of the P-PMOs tested against JUNV (CL-trm, LS-trm andSS-trm, SEQ ID NOS:2-4, respectively) were directed at the conservedgenomic 5′-termini. To determine the effectiveness of the same P-PMOsagainst a distantly related Old World arenavirus, lymphocyticchoriomeningitis virus (LCMV) was used in experiments identical to thosedescribed in Example 1. P-PMOs directed at the conserved termini(CL-trm, LS-trm and SS-trm, SEQ ID NOS:2-4, respectively) stronglysuppressed LCMV proliferation, reducing viral titers by 100 to100000-fold as shown in FIG. 8. The same negative controls were used asdescribed in Examples 1 and 2, a scramble control sequence (DSCR) anduntreated, and demonstrated no inhibition of LCMV replication as shownin FIG. 8. P-PMOs directed to initiation codons and other sequences notconserved among arenaviruses were ineffective at reducing LCMVproliferation (i.e., LS-AUG, CL-AUG, SS-AUG, CS-FP3 and SS-IG). Theseresults support the claim that P-PMOs directed to the genomic terminiwill have a high probability of inhibiting any strain of arenavirus. TheLCMV-Armstrong strain used in these experiments is known to have a twobase pair mismatch with each of the P-PMOs that showed antiviralactivity, CL-trm, LS-trm and SS-trm, SEQ ID NOS:2-4, respectively.

Certain non-standard nucleotide analogs such as inosine can be includedin antisense oligomers to permit binding to target sequences that areheterogeneous at specific sites, as is the case with the arenavirusterminal sequence (e.g., see FIG. 3). Exemplary targeting oligomers thatutilize this approach are listed as SEQ ID NOS:5 and 6.

Sequence Listing Name SEQ ID NO Target Sequences (5′ to 3′) 1 CGC ACMGDG GAT CCT AGG C Oligomer Targeting Sequences (5′ to 3′) CL-trm CGC CTAGGA TCC CCG GTG CG 2 LS-trm CGC CTA GGA TCC CCG GTG CGC 3 SS-trm GCC TAGGAT CCA CTG TGC GC 4 PanCL GCC TAG GAT CCI CIG TGC G 5 PanLS CGC CTA GGATCC ICI GTG CG 6 Peptide Sequences (NH₂ to COOH) P003 RRRRRRRRRAhxβAla 7P007 (RAhxR)₄AhxβAla 8 P008 (RAhx)₈βAla 9 RX4 (RAhx)₄βAla 10  RXR2(RAhxR)₂AhxβAla 11  RXR3 (RAhxR)₃AhxβAla 12 

1-17. (canceled)
 18. A method of inhibiting viral infection in mammaliancells by an Arenavirus in the Arenaviridae family, comprising (a)exposing the cells to an antisense oligonucleotide compound capable ofuptake by mammalian host cells, wherein the antisense oligonucleotidecompound is comprised of morpholino subunits and phosphorous-containingintersubunit linkages having the following structure (I):

wherein: Y₁=O; Z=O; Pi and Pj are independently a purine or pyrimidinebase-pairing moiety effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide; X is alkyl, alkoxy, thioalkoxy,amino, alkyl amino, or dialkylamino when the phosphorous-containingintersubunit linkage is uncharged at physiological pH; and X ispiperazynyl when the phosphorous-containing intersubunit linkage ischarged at physiological pH, wherein the antisense oligonucleotidecompound: (i) comprises at least one phosphorous-containing intersubunitlinkage which is charged at physiological pH; (ii) contains between12-40 base-pairing moieties; (iii) comprises a targeting sequence of atleast 12 contiguous base-pairing moieties complementary to SEQ ID NO:1;and (iv) optionally comprises a hydrophilic polymer for enhancing thesolubility of the antisense oligonucleotide compound; and (b) by saidexposing, forming a heteroduplex structure composed of the virus' vRNAor vcRNA strand and the antisense oligonucleotide compound, wherein theheteroduplex structure has a Tm of dissociation of at least 45° C. 19.The method of claim 18, wherein X is dimethylamino for thephosphorous-containing intersubunit linkages which are uncharged atphysiological pH.
 20. The method of claim 18, wherein at least 80% ofthe phosphorous-containing intersubunit linkages are uncharged atphysiological pH.
 21. The method of claim 18, wherein about onephosphorous-containing intersubunit linkage is charged at physiologicalpH per every five phosphorous-containing intersubunit linkages which areuncharged at physiological pH.
 22. The method of claim 18, wherein twoof the phosphorous-containing intersubunit linkages are charged atphysiological pH.
 23. The method of claim 18, wherein the antisenseoligonucleotide compound contains between 14-24 base-pairing moieties.24. The method of claim 23, wherein the antisense oligonucleotidecompound contains 20 base-pairing moieties.
 25. The method of claim 18,wherein the antisense oligonucleotide compound comprises a targetingsequence of 19 contiguous base-pairing moieties complementary to SEQ IDNO:1.
 26. The method of claim 18, wherein the antisense oligonucleotidecompound comprises a polyethyleneglycol moiety for enhancing thesolubility of the antisense oligonucleotide compound.
 27. The method ofclaim 18, for use in treating a mammalian subject infected by anArenavirus in the Arenaviridae family, wherein said exposing includesadministering to the subject, a pharmaceutically effective amount of theantisense oligonucleotide compound, and which further includescontinuing said administering until a significant reduction in viralinfection or the symptoms thereof is observed.
 28. The method of claim27, further comprising administering a second anti-viral compound to thesubject.
 29. The method of claim 18, for use in treating a mammaliansubject at risk of infection by an Arenavirus in the Arenaviridaefamily, wherein said exposing includes administering to the subject, anamount of the oligonucleotide compound effective to inhibit infection ofsubject host cells by the virus.
 30. An antisense oligonucleotidecompound comprised of morpholino subunits and phosphorous-containingintersubunit linkages having the following structure (I):

wherein: Y₁=O; Z=O; Pi and Pj are independently a purine or pyrimidinebase-pairing moiety effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide; X is alkyl, alkoxy, thioalkoxy,amino, alkyl amino, or dialkylamino when the phosphorous-containingintersubunit linkage is uncharged at physiological pH; and X ispiperazynyl when the phosphorous-containing intersubunit linkage ischarged at physiological pH, wherein the antisense oligonucleotidecompound: (i) comprises at least one phosphorous-containing intersubunitlinkage which is charged at physiological pH; (ii) contains between12-40 base-pairing moieties; (iii) comprises a targeting sequence of atleast 12 contiguous base-pairing moieties complementary to SEQ ID NO:1;and (iv) optionally comprises a hydrophilic polymer for enhancing thesolubility of the antisense oligonucleotide compound; and wherein theantisense oligonucleotide compound is capable of binding to vRNA/vcRNAor mRNA strands of an Arenavirus in the Arenaviridae family to form aheteroduplex structure having by a Tm of dissociation of at least 45° C.31. The antisense oligonucleotide compound of claim 30, wherein X isdimethylamino for the phosphorous-containing intersubunit linkages whichare uncharged at physiological pH.
 32. The antisense oligonucleotidecompound of claim 30, wherein at least 80% of the phosphorous-containingintersubunit linkages are uncharged at physiological pH.
 33. Theantisense oligonucleotide compound of claim 30, wherein about onephosphorous-containing intersubunit linkage is charged at physiologicalpH per every five phosphorous-containing intersubunit linkages which areuncharged at physiological pH.
 34. The antisense oligonucleotidecompound of claim 30, wherein two of the phosphorous-containingintersubunit linkages are charged at physiological pH.
 35. The antisenseoligonucleotide compound of claim 30, wherein the antisenseoligonucleotide compound contains between 14-24 base-pairing moieties.36. The antisense oligonucleotide compound of claim 30, wherein theantisense oligonucleotide compound comprises a targeting sequence of 19contiguous base-pairing moieties complementary to SEQ ID NO:1.
 37. Theantisense oligonucleotide compound of claim 30, wherein the antisenseoligonucleotide compound comprises a polyethyleneglycol moiety forenhancing the solubility of the antisense oligonucleotide compound.